TfL concluded that the minicab app is “not fit and proper” to operate in the capital due to concerns which have “public safety and security implications”.
These include its approach to reporting serious criminal offences and how it carries out background checks on its drivers.
Uber was given just a four-month temporary licence in May.
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Mayor of London Sadiq Khan said in a statement: “I want London to be at the forefront of innovation and new technology and to be a natural home for exciting new companies that help Londoners by providing a better and more affordable service.
“However, all companies in London must play by the rules and adhere to the high standards we expect - particularly when it comes to the safety of customers. Providing an innovative service must not be at the expense of customer safety and security.
“I fully support TfL’s decision - it would be wrong if TfL continued to license Uber if there is any way that this could pose a threat to Londoners’ safety and security.
“Any operator of private hire services in London needs to play by the rules.”
There had been growing speculation that the app could be banned from London.
Opponents of the firm claim it causes gridlocked roads and does not do enough to regulate its drivers.
Uber enables users to book cars using their smartphones, and is available in cities across the UK.
Some 3.5 million passengers and 40,000 drivers use the Uber app in London.
Last month Uber was accused by police of allowing a driver who sexually assaulted a passenger to strike again by not reporting the attack, along with other serious crimes.
In a strongly worded letter, Inspector Neil Billany of the Metropolitan Police’s taxi and private hire team suggested the company was putting concerns for its reputation over public safety.
He cited the case of a man who worked for Uber being allowed to stay on the books despite an allegation of sexual assault, leading to another “more serious” attack on a woman in his car.
A string of serious crimes it allegedly failed to report included more sexual assaults and an incident in which a driver produced what was thought to be pepper spray during a road-rage argument.
Uber said at the time it was “surprised by this letter” and claimed it does not reflect the “good working relationship we have with the police”.
Steve McNamara, general secretary of the Licensed Taxi Drivers’ Association said: “The Mayor has made the right call not to relicense Uber. Since it first came onto our streets Uber has broken the law, exploited its drivers and refused to take responsibility for the safety of passengers.
“We expect Uber will again embark on a spurious legal challenge against the Mayor and TfL, and we will urge the court to uphold this decision. This immoral company has no place on London’s streets.”
Maria Ludkin, legal director of the GMB union, which took Uber to an employment tribunal last year over workers’ rights, said: “This historic decision is a victory for GMB’s campaign to ensure drivers are given the rights they are entitled to - and that the public, drivers and passengers are kept safe.
“As a result of sustained pressure from drivers and the public, Uber has suffered yet another defeat - losing its license to operate in London.
“It’s about time the company faced up to the huge consequences of GMB’s landmark employment tribunal victory - and changed its ways.
“No company can be behave like it’s above the law, and that includes Uber. No doubt other major cities will be looking at this decision and considering Uber’s future on their own streets.”
And Mick Cash, general secretary of the Rail, Maritime and Transport union, said: “Uber has consistently failed to reach acceptable standards of service, safety and security and we applaud this decision which is a victory for passengers and also a vital step in protecting the livelihoods of the skilled and experienced London taxi drivers who are being unfairly undercut by Uber.
“This is a success for our campaigning and all those who work in the trade and must be a stepping stone to end the deregulation in the industry which has created such chaotic, unsafe and exploitative conditions.
“The next steps should include the introduction of a statutory definition of plying for hire and also for MPs to get behind the Private Members Bill put forward by Daniel Zeichner MP to reform the taxi and private hire industry.”
TfL said Uber is allowed to launch an appeal against the decision within 21 days and can continue operating “until any appeal processes have been exhausted”.
It added: “No further comment will be made by TfL pending any appeal of this decision.”
Uber has vowed to appeal after Transport for London said it will not be issued with a new licence and was “not fit and proper” to operate in the capital.
TfL said it took the decision on the grounds of “public safety and security implications”.
But Uber, which is used by 3.5 million people and 40,000 drivers in London, hit back, saying it would appeal and claiming the move “would show the world that, far from being open, London is closed to innovative companies”.